Staining was performed according to standard protocols using the following antibodies: Bmi1 (1:100, 05–637, Merck Millipore), Ring1b (1:400, NBP1–49966, Novus Biologicals), E-Cadherin (1:100, 3195, Cell Signaling Technology), Gata6 (1:100, 22600, Abcam) and H2AK119ub (1:400, 8240, Cell Signaling Technology).
Gata6
Manufactured by Abcam
Sourced in United States, United Kingdom
GATA6 is a transcription factor that plays a role in the regulation of gene expression. It is involved in the development and differentiation of various cell types, including those in the cardiovascular, respiratory, and endodermal systems. GATA6 is used in research applications to study gene expression and cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Ecl kit, by Thermo Fisher Scientific (4 mentions)
Gata4, by Abcam (2 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (2 mentions)
C caspase 3, by Cell Signaling Technology (2 mentions)
Difco skim milk, by BD (2 mentions)
Genegnome5, by Syngene (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
H2ak119ub, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
β catenin, by Proteintech (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
D9542, by Merck Group (1 mentions)
Sp8 laser scanning confocal microscope, by Leica camera (1 mentions)
Ripa lysis containing proteinase inhibitors, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bca kit, by Beyotime (1 mentions)
9 protocols using gata6
1
Immunostaining of Stem Cell Markers
2
Immunohistochemical Analysis of EMT Markers in CCA
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The expression of GATA6, MUC1, β-catenin and EMT markers (E-cadherin, N-cadherin and vimentin) in CCA samples was detected using IHC. IHC was performed using the method described in our previous study14 (link). Antibodies against GATA6 (1 : 200, Abcam, Cambridge, MA, USA), MUC1 (1 : 500, Abcam, Cambridge, MA, USA), β-catenin (1 : 100, Proteintech, Wuhan, China), E-cadherin (1 : 100, Proteintech, Wuhan, China), N-cadherin (1 : 100, Proteintech, Wuhan, China) and vimentin (1 : 100, Proteintech, Wuhan, China) were used. Two independent investigators assessed the percentage and staining intensity of the positive cancerous cells.
3
Immunofluorescence Staining of ESCs
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ESCs or ESC-derived cells were collected and fixed with 4% paraformaldehyde for half an hour at room temperature. Then, the cells were washed with PBST (phosphate-buffered saline, 0.1% Triton X-100) for three times, each for 15 min. Following the incubation with blocking buffer (5% normal horse serum, 0.1% Triton X-100, in PBS) for 2 h at room temperature, the cells were incubated with primary antibodies at 4 °C overnight. Antibodies used were POGZ (Abcam, ab171934), OCT4 (Proteintech, 60,242–1-Ig), NANOG (Bethyl, A300-397A), GATA6 (Abcam, ab175349), HP1 (Abcam, ab213167), and PH3 (CST, #9701). After three times of wash with PBST, the cells were incubated with secondary antibodies (1:500 dilution in blocking buffer, Alexa Fluor 488, Life Technologies) at room temperature for 1 h in the dark. The nuclei were counterstained with DAPI (Sigma, D9542, 1:1000). After washing with PBS for twice, the slides were mounted with 100% glycerol on histological slides. Images were taken by a Leica SP8 laser scanning confocal microscope (Wetzlar, Germany). About 10 images were taken for each slide. Quantification of IF staining was performed using ImageJ [30 (link)].
4
Western Blot Analysis of GATA6 Expression
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After transfection for 48 h, RIPA lysis containing proteinase inhibitors (Beyotime Institute of Biotechnology, Haimen, China) was used to extract total protein. The protein concentrations were tested with the BCA kit (Beyotime Institute of Biotechnology). The total proteins (50 µg) were added into the well with SDS-PAGE and electrophoresis at 60 V was performed when bromophenol blue ran out of the bottom. The proteins were then transferred to nitrocellulose filter (NC) membranes, and skimmed milk (5–10%) was used to block he membranes at room temperature for 2 h. Subsequently, primary antibodies (GATA6, Abcam, Cambridge, UK; GAPDH, Cell Signaling Technology, Inc., Danvers, MA, USA) were added in to incubate the samples at 4°C overnight. After being washed with 1X TBST (pH 7.4) three times, the secondary antibodies were added in and incubated at room temperature for 2 h. Protein bands were detected using the chemiluminescence method (ECL; Millipore Billerica, MA, USA). GAPDH served as a loading control.
5
Autophagy Regulation in Fibroblast Cells
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DHA, colchicine, PDGF-BB, Etoposide, CQ, rapamycin (Rapa), 3-MA, bafilomycin A1, dimethyl sulfoxide (DMSO), anti-rabbit IgG, and anti-mouse IgG were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified essential medium (DMEM), Opti MEM medium, phosphate-buffered saline (PBS), trypsin-EDTA and fetal bovine serum (FBS) were bought from GIBCO BRL (Grand Island, NY, USA). Primary antibodies against p53, p16, p21, α-SMA, Hmga1, LC3-I/II, ULK1, p-ULK1, mTOR, p-mTOR, Atg3, Atg5-Atg12, Atg6, Atg7, Atg14, p62, β-galactosidase and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibody against α1(I) procollagen was purchased from Epitomics (San Francisco, CA, USA). Primary antibodies against Ki67, p62 and GATA6 were purchased from Abcam Technology (Abcam, Cambridge, UK). Atg5 siRNA, GATA6 siRNA, negative control siRNA, Atg5 plasmid, GATA6 plasmid, negative control vectors and mRFP-GFP-LC3 plasmid were purchased from Hanbio (Shanghai, China). MegaTran 1.0 transfection reagent was from OriGene (Rockville, MD, USA).
6
Comprehensive Protein Analysis Protocol
7
Chick Embryo Molecular Analysis
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HH4, HH7 and HH10 chick embryos were collected and performed as pervious reported [21] . Antibodies: E-cadherin (BD Transduction Laboratories, USA), N-cadherin (DSHB, USA), Slug (DSHB, USA), Mef2c (Abcam, USA); GATA4 (Abcam, USA); TBX5 (Abcam, USA); GATA6 (Abcam, USA); β-catenin (Abcam, USA); PCNA (Santa Cruz Biotechnology, USA); C-caspase3 (Cell Signaling, USA); HRP-conjugated anti-mouse IgG, HRP-conjugated anti-rabbit IgG (Cell Signaling Technology, USA).
8
Chick Embryo Protein Expression Analysis
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Chick embryos (HH4 and HH7) were collected and lysed with CytoBuster™ Protein Extraction Reagent (#71009, Novagen). The total protein concentration was established using a BCA quantification kit (BCA01, DingGuo BioTECH, CHN).
Samples containing equal amounts of protein were resolved by SDS-PAGE and then transferred to PVDF membranes (Bio-Rad). The membranes were blocked with 5% Difco™ skim milk (BD) and then incubated with primary and secondary antibodies.
The antibodies used were TBX5 ， GATA4 and GATA6 (Abcam USA), HRP-conjugated anti-mouse IgG and anti-rabbit IgG (Cell Signaling Technology, USA). All primary and secondary antibodies used were diluted to 1:1000 and 1:2000 in 5% skim milk, respectively. The protein bands of interest were visualized using an ECL kit (#34079, Thermo Fischer Scientific Inc.) and GeneGnome5 (Syngene). The staining intensity of the bands was determined and analyzed using Quantity One software (Bio-Rad).
Samples containing equal amounts of protein were resolved by SDS-PAGE and then transferred to PVDF membranes (Bio-Rad). The membranes were blocked with 5% Difco™ skim milk (BD) and then incubated with primary and secondary antibodies.
The antibodies used were TBX5 ， GATA4 and GATA6 (Abcam USA), HRP-conjugated anti-mouse IgG and anti-rabbit IgG (Cell Signaling Technology, USA). All primary and secondary antibodies used were diluted to 1:1000 and 1:2000 in 5% skim milk, respectively. The protein bands of interest were visualized using an ECL kit (#34079, Thermo Fischer Scientific Inc.) and GeneGnome5 (Syngene). The staining intensity of the bands was determined and analyzed using Quantity One software (Bio-Rad).
9
Protein Expression Analysis in Chick Embryos
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HH10 chick embryos were collected and lysed with CytoBuster™ Protein Extraction Reagent (#71009, Novagen). Total protein concentrations were assessed via a BCA quantification kit (BCA01, DingGuoBioTECH, CHN). Samples containing identical amounts of protein were fractionated by SDS-PAGE, and then transferred to PVDF membranes (Bio-Rad). Membranes were blocked with 5% Difco™ skim milk (BD) and subsequently incubated with primary and secondary antibodies, then bands of interest protein were visualized using the ECL kit (#34079,Thermo) and GeneGnome5 (SYNGENE). Gray scale of bands was analyzed using Quantity One software (Bio-Rad).Antibodies: VEGFR2 (Abcam, USA); GATA4 (Abcam, USA); TBX5 (Abcam, USA); GATA6 (Abcam, USA); PCNA (Santa Cruz Biotechnology, USA); c-Caspase3 (Cell Signaling, USA); HRP-conjugated anti-mouse IgG, HRPconjugated anti-rabbit IgG (Cell Signaling Technology, USA). All primary antibodies were diluted 1000-fold in 5% skim milk, and secondary antibodies were diluted 2000fold.
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